Method for the preparation of anallergic probiotic bacterial cultures and related use

ABSTRACT

The aim of the present invention is a method for the preparation of anallergic probiotic bacterial cultures.

The aim of the present invention is a method for the preparation ofanallergic probiotic bacterial cultures.

It is known that the human gastrointestinal (GI) tract contains acomplex microbial, community, called microbiota, mainly consisting ofstrictly anaerobic bacteria capable of playing different actions witheffects which affect the local gastrointestinal level and indirectly thegeneral systemic level, involving almost all the organs and the hostfunctions.

The intestinal microflora, consisting of a large variety of differentspecies (400-500) capable to colonize both the intestinal mucosa and theparticles of ingested food, is then able to strongly condition thehealth of the individual.

The composition of the intestinal microflora can be altered by severalfactors, such as the age, the physiological condition of the individual,the presence of different pathologies, the stress and above all thediet.

As a consequence of endogenous and exogenous negative factors, there isa decrease of the useful bacteria (typically bacteria belonging to thelactic acid group in the small intestine and bifidobacteria in the largeintestine) and an increase of pathogenic enterobacteria, streptococciand clostridia.

The administration of specific probiotics through medicinal specialties,dietetics, integrators and above all food (so-called “functional” or“nutraceutic” foods) allows to re-equilibrate the microflora of thehost, by restoring an optimal intestinal functionality.

The term “probiotic” commonly relates to living microorganisms, selectedfrom the intestinal microflora of healthy individuals, which onceadministered in opportune quantities and for an adequate time, are ableto colonize, also if only temporarily, the different tracts of theintestine and to impart beneficial effect to the health of the hostorganism.

Belonging to the probiotics are mainly some species of the generaLactobacillus, Bifidobacterium, Streptococcus, Pediococcus, Lactococcus,Propionibacterium, Leuconostoc and, in a lower extent, Saccharomyces,Bacillus and Enterococcus.

Between the healthy and beneficial effects, shown by severe clinicaltests carried out all over the world and induced in the consumer by theintake of probiotics there can be mentioned:

1. Stimulation of the immune system;

2. Induction of antimutagenic and antigenotoxic effects;

3. Antitumoral and antimetastatic action referring, for example, to:

large intestine carcinoma

breast carcinoma

bladder cancer

4. Iprovement in the nutrients absorption;

5. Decrease of the lactose intolerance symptoms;

6. Improvement of the intestinal motility due to the lowering of the pHand decrease of the constipation;

7. Decrease of the absorption of cholesterol and fats;

8. Antidiarrhoic, anti-hypertensive, anti-diabetic activities inhibitinginfections of the female urogenital system;

9. Prevention activity of geriatric pathologies.

The probiotics for oral use must be characterized by the followinggeneral and functional requirements:

i) human intestinal origin; from individuals in good health conditions;

ii) bio-safety; they must not cause side effects especially in weakenedor immune depressed persons;

iii) Resistance and viability; they must have such resistance featuresto be able to survive the gastric juice, the pancreatic and bilesecretions so as to reach the ileum and the colon in an undamaged wayand still perfectly viable.

The competition which begins between probiotic microorganisms andpathogenic microflora can only occur if, once ingested, the probioticmicroorganisms are able to reach the intestinal tract and therefore tosurvive the gastric acidity and the high concentration of the bilesalts. Generally, once they have reached the intestinal tract, they aresaid to begin through adhesion mechanisms which involve proteins and/orcarbohydrates with specific adhesion functionalities to the intestinalvilli.

At industrial level, the probiotics are produced in form of freeze driedbacterial culture, namely the growth of the cells in a proper medium isdetermined (fermentation) and next, following to concentration andpurification of the biomass, the dehydration of the same is performed byfreeze drying. Such process is required for allowing the bacteria toreproduce themselves in an adequate number (fermentative step) and topreserve themselves for a long time (freeze drying step).

In order that the fermentative process is successfully carried out andwith adequate yields, it is necessary to provide the cells with carbonand nitrogen sources, oligo-elements and bioactivators in opportunequantities.

Traditionally, dealing with commonly said “lactic” bacteria, theselected substrates used as a nitrogenous source are serum, milkserum-proteins, hydrolyzates and casein peptones, caseinates, etc.,while as a carbon source, lactose and glucose are usually employed, asthey can easily be metabolized from almost all the living organisms.

In the last years, probably also because of a diet too little varied andtoo rich in proteins and lipids, in Europe and Western countries asensitive increase of the number of persons suffering from allergic-typepathologies has been registered.

The immune reactions of type IV or IgE-mediated are called “allergic”,during which, following to a first sensitizing contact (which can occurin any time of the life, also the intrauterine one) specific IgEs areproduced with a histamine-mediated mechanism.

Such reactions can be also caused by extremely reduced doses ofmolecules called “allergens”, with clinical consequences which canchange from simple slight skin reactions to anaphylactic shock and dead.

Due to the dangerousness of these substances for some people, theEuropean Community has adopted a rule such that in the label “the use inthe production and the presence in the food” of foods belonging to thefollowing classes (Annex III bis of the above instruction) has to beclearly shown (art. 6 sub. 10, instruction 2000/13/EC thus modified bythe instruction 2003/89/EC):

gluten-containing cereals (that is wheat, rye, barley, oat, spelt, kamutor their hybridized-strains) and derived products; crustacea andcrustacea-based products; eggs and eggs-based products; fish andfish-based products; peanuts and peanut-based products; soy andsoy-based products; milk and milk-based products (including thelactose); fruits with shell, that is almonds (Amigdalus communis L.),hazelnuts (Corylus avellana), common walnuts (Juglans regia), acagiuwalnuts (Western Anacardium), pecan walnuts [Carya illinoiesis (Wangenh)K. Koch], Brazil walnuts (Bertholletia excelsa), pistachio nuts(Pistacia vera), Queensland walnuts (Macadamia ternifolia) and derivedproducts; celery and celery-based products; mustard and mustard-basedproducts; sesame seeds and sesame seeds-based products; sulfur dioxideand sulphites in concentrations higher than 10 mg/kg or 10 mg/lexpressed as SO₂.

As, typically for the production of probiotics, substrates based on milk(as a nitrogenous source) and glucose (as a carbon source), which isusually originated from the starch (also wheat starch), derivatives areused, the probiotics can be considered dangerous if administered toparticularly sensitive subjects, even if, at the most, they actuallycould contain only small traces of allergens (therefore derived frommilk and/or gluten).

In case of milk, there can be individualized two causes of adversereaction to this food; the cow's milk proteins allergy (APLV) and thelactose intolerance.

The milk, together with the egg, is the more allergising food; thisfeature thereof is determined by the proteinic substances contained bythe same, that is mainly alfa-lactoalbumin, beta-lactoglobulin andcaseins.

The allergy to the cow's milk proteins is a relatively frequentpathology in the first year of life. The symptomatology is, in the50-70% of the cases, of a gastroenteric type, yet in the 50-70% of thecases there are skin disorders, in the 20-30% respiratory disorders andin the 5-9% systemic disorders (anaphylaxis).

The APLV tends to fade itself after the first year of life and todisappear towards the 10 years and is unusual in the adults.

An opposite course has the lactose intolerance, which is very unusual inthe first year of life and very frequent in the adult, in particular insome populations (African, Asiatic, American Indian).

The milk is a fundamental food since the birth and the new-born alreadyproduces the enzyme necessary for the fission of the milk sugar, thelactose, in its simple components glucose and galactose. After the firstyear of life, the milk becomes a less important food and the lactase isspontaneously reduced, such that many adults become intolerant (notallergic) to the milk. The celiac disease is an enterophatic, chronic,immune-mediated inflammatory disease, risen from the ingestion ofgluten, a “storage” protein naturally contained in some cereals.

This food intolerance affects genetically predisposed persons, having animmune system which responds in an abnormal way to the ingestion ofproteinic fractions typical of the wheat gluten, spelt (a kind ofwheat), kamut and spella (a kind of wheat), barley, rye, triticale (across between wheat and rye) and their derivatives. Some individualsalso present intolerance to the oat proteins.

Technically the term “gluten” applies for the combination of the simpleprolaminic (rich in proline), called “gliadins”, and glutelinic (rich inglutamine), called “glutenins”, proteins of the cereals above mentioned.

In the context of the celiac disease, the term “gluten” is often usedwith reference to all kinds of proteins contained in the cereals, evenif between the different proteinic fractions which form the gluten, thegliadin seems to be the most detrimental.

In the people affected by the celiac disease, the assumption of gluten,also in small quantities, is capable of causing the abnormal response ofthe immune system: the transglutaminase, an enzyme existing in theintestinal mucosal tissue, binds to the gliadin and through deamidationtransforms it in a molecule capable of activating the T cells (cells ofthe immune system capable of mediating all the immune responses towardsthe proteinic antigens), with a consequent production ofanti-transglutaminase IgG and IgA and anti-endomise IgA immunoglobulins.

In a first stage, an increase of the intraepithelial intestinalactivated T cells occurs, while with the progress of the disease theincrease relates both to the lymphocytes and the infiltrated plasmacells of the own lamina, with a production of metalloproteinasesresponsible for the shortening of the villi and therefore the damage tothe intestinal mucosa.

The possibility of preventing the development or treating the celiacdisease does not exist so far, therefore a gluten-free diet, strictlycarried out for all the lifetime is the only therapy capable of ensuringto the people affected by the celiac disease a perfect health.

The celiac persons must eliminate also the smallest traces of flour ofthe dangerous cereals, because the intake of gluten, also in minimalquantities, can unbridle the autoimmune response.

As the ratio of the quantity of ingested gluten to the toxic effectinduced at the intestinal level has still not been defined, the term“traces” has a fundamental practical importance in the treatment of theceliac disease and implications on the food legislation plan, because itis related at the maximum limit of “acceptable” gluten (threshold) inthe products suitable for the diet of the celiac person. All the skilledpersons agree that a dose of 100 mg per day of gliadin, equal to 200 mgof gluten, i.e. about 3 g of bread, is sufficient to cause, in most ofthe celiac persons, the increase of the intestinal intraepitheliallymphocytes, a premature sign of persistent intestinal inflammation.

With reference to the minimum threshold, the few scientific workscarried out so far would seem to show that the ingestion up to 10milligrams per day of gliadin (equal to 20 parts per million of gluten)is not capable of sensitively damaging the intestinal mucosa, but candetermine, in a minority of the cases, the gastroenteric symptomsoccurrence.

At the international legislative level, the old standard “Codex Standardfor Gluten-Free Foods”, still in force, shows, as the maximum glutencontent in the diet-therapeutic products, 0.05 of nitrogen per 100 g ofdry product (with reference to the wheat starch), which corresponds to agluten fraction equal to about 500 ppm.

However, a right and proper review of the aforesaid guide line is takingplace, which seems to foresee that the gluten-free foods resulting fromnaturally gluten-free ingredients have not to contain more than 20 ppmof gluten, while the gluten-free foods, deriving from cereals withgluten, can have a maximum limit of 200 ppm of gluten.

In France, Great Britain and the Netherlands, waiting for the review ofthe aforesaid guide line, they consider, as the maximum limit for thegluten-free products, 200 ppm.

In the national ambit, the current rule seems to be more precautionarythan the community and international one, in fact a limit of “20 partsper million”, both for the foods manufactured with raw materialsnaturally free from gluten and the foods purified from such substance,is established.

The rule foresees that “if, in the composition of the food product or inthat of one or more ingredients (flavourings, additives or co-adjuvants)which form the same, are present cereals containing gluten or substancesderiving therefrom and/or if from the productive process a quantity ofgluten can derive in the end product, analytically determined as higherthan 20 parts per million, such product will have to show in the label,at the foot of the ingredients list and in a well visible way, the words“gluten-containing product”.

The complete exclusion of the gluten from the diet is not however easyto carry out, considering that cross-contamination phenomena of cerealsand derivatives (starches, flours, starch flour, etc.) naturally freefrom gluten, already at the milling industry level, can occur.

Such products are then used by the food industry in the preparation ofcomplex foods based on multiple technological ingredients, additives andco-adjuvants of different origin and nature.

A recent research has shown that up to 6% of the “theoretically”gluten-free products, based on the reported ingredients, actuallycontain over 30 mg of gliadin per 100 g of end product, equal to 600 ppmof gluten.

For the purpose of excluding a possible gluten-contamination, it istherefore necessary to consider, for each commercialized food product,not only all the ingredients used and the processing, but also all theproductive chain of each single ingredient.

Therefore, there remains the need of being able to produce probioticbacterial cultures free from allergising substances due to the use offermentative substrates based on milk or cereals or, alternatively, dueto unintentional or cross-contaminations. In fact, it is neverthelesspossible that, due to cross-, unintentional and accidentalcontaminations, some components used in the productive process bringsallergens traces.

Therefore, there remains the need of being able to provide a method forthe preparation of probiotic bacterial cultures which foresees, in eachstep of the productive process, including the fermentation, the use ofanallergic substances. In particular, it is desirable to locate andselect fermentative substrates, different from milk and its derivativesand the gluten-containing cereals, which represent a good nitrogen andcarbon source.

Therefore, all the sector operators agree that, so far, there remains avery strong need of providing a method for the preparation of probioticbacterial cultures capable of using substrates alternative to those useduntil today and, simultaneously, capable of reducing the cross-,unintentional and accidental contaminations, should they occur.

In particular, there remains the need of providing a method for thepreparation of probiotic bacterial cultures which foresees a doublelevel of safety relating to the absence of allergising substances.

An aim of the present invention is to provide a method for thepreparation of culture media capable of overcoming the limits of theknown art.

Another aim of the present invention is to provide a methodology for theproduction of probiotic bacterial cultures safe to administer to all thepopulation, also to the people affected by allergies.

These and other aims, which will result apparent from the followingdetailed description, have been attained by the Applicant, which hasimproved a methodology which includes a double safety level relating tothe absence of allergens in the productive processes of probioticbacterial cultures.

In particular, the Applicant has set up a production methodology inwhich selected anallergic fermentation substrates (anallergic rawmaterials) are used, capable of ensuring a proper nitrogen and carbonsource to the probiotic cultures.

A method for the preparation of an anallergic probiotic bacterialcultures, a composition including said culture and the use of saidculture for the preparation of so-called “functional” or “nutraceutic”foods form the subject of the present invention, having the features asdefined in the appended claims.

In an embodiment of the invention, the strains of said bacterial culturebelong to the genera: Lactobacillus, Bifidobacterium, Streptococcus,Pediococcus, Lactococcus, Propionibacterium, Bacillus, Saccharomyces,Enterococcus, Leuconostoc.

Preferably, of the genus Lactobacillus, the following species have founduse: L. pentosus, L. plantarum, L. casei, L. casei ssp. paracasei, L.casei ssp. rhamnosus, L. acidophilus, L. delbrueckii ssp. bulgaricus, L.fermentum, L. gasseri.

Examples of used strains of said species are reported in the enclosedTable 1. Preferably, of the genus Bifidobacterium, the following specieshave found use: B. longum. B. breve, B. lactis, B. adolescentis and B.pseudocatenulatum.

Examples of used strains of said species are reported in the enclosedTable 1. Preferably, of the genus Lactococcus the following species havefound use: L. lactis and L. lactis ssp. Lactis.

Examples of used strains of said species are reported in the enclosedTable 1. Preferably, of the genus Streptococcus the following specieshave found use: S. thermophilus.

Examples of used strains of said species are reported in the enclosedTable 1.

In a particularly preferred embodiment of the invention, the bacteria ofsaid bacterial culture are selected from the group including theprobiotic bacterial strains reported in the enclosed Table 1.

The enclosed Table 1 identifies strains deposited at the BCCM/LMGBacteria Collection of Gent, Belgium and at the DSMZ-Deutsche Sammlungvon Mikroorganismen and Zellkulturen GmbH, Inhoffenstrasse 7 B, 38124Braunschweig, GERMANY; the deposits are in accordance with the BudapestTreaty.

The Applicant has found useful to select and employ particularanallergic raw materials. In particular, the Applicant has found, as anitrogen source, peptones and/or proteinic hydrolyzates of vegetaland/or animal origin, naturally free from gluten and allergens of milkyorigin and, as a carbon source, glucose and/or other mono- ordisaccharides derived from the hydrolysis of more complexpolysaccharides typical of vegetal species naturally free from glutenand allergens of milky origin.

The peptones of vegetal origin are selected from the group including:rice, potato, maize, chestnuts, tapioca, manioca, pea, broad beans andtheir mixtures however capable of promoting the fermentation bacterialgrowth, but without producing allergens, either of milky nor glutentypes.

In a first preferred embodiment, the method subject of the presentinvention foresees the use, as a nitrogen source, of one or morepeptones and/or anallergic proteinic hydrolyzates and, as a carbonsource, glucose and/or other mono- or disaccharides derived fromhydrolysis of complex polysaccharide (anallergic raw materials).

In a second preferred embodiment, the method subject of the presentinvention foresees a pre-treatment of the raw materials with enzymessuitable for the removal of traces, if any, of allergens deriving fromcross-contamination occurred along the productive and/or distributivechain.

In the context of the present invention, the culture substrate is ananallergic culture substrate of vegetal and/or animal origin, naturallyfree from gluten, allergens or milky origin and all the substancesbelonging to the list of the annex III bis of the instructions(anallergic raw materials) above mentioned. The use of anallergic rawmaterials above mentioned allows to obtain certifiable probiotics forthe use in allergic persons, as the non-use of substances belonging tothe list of the annex III bis of the aforesaid community instructionsand the use of ingredients certified from the supplier as free from suchsubstances can be assured.

Then, by virtue of the fact that the absence of any chemical substancesin a given sample is not scientifically demonstrable, but one can simplydetermine that the quantity possibly existing is lower than thedetection limit of the analytical method used (even if the moresensitive and refined method known in the art was used), also the use ofan enzymatic pre-treatment results to be a source of an additionalguarantee.

By mere way of example, some anallergic formulations of medium for thegrowth of probiotic bacterial cultures are reported below.

The components of a culture medium must bring nitrogen sources (in thiscase the peptones and/or proteinic hydrolyzates), carbon sources (inthis case, the glucose and/or other mono- or disaccharides derived fromhydrolysis of complex polysaccharides), growth bioactivators andvitamins (in this case from yeast extract) and mineral salts.

For example, a culture medium can contain:

glucose preferably selected from: maize starch, potato, beet sucrose orcane sucrose;

peptone preferably selected from: rice, potato, maize, chestnut,tapioca, maniocak pea, broad beans, bean or generally legumes and theirmixtures;

peptone preferably selected from: meat;

yeast extract; mineral salts (such as, by mere way of example: acetates,carbonates, phosphates, hydrogen phosphates, chlorides, citrates,sulfates and others); builder (if necessary, such as: Tween, lecithinsand other) and drinking water.

One of the formulation suitable for the growth of strains of theLactobacillus and Bifidobacterium genera could preferably include thefollowing ingredients:

glucose (from the sources above listed) 10-100 g/l rice peptone 10-50g/l meat peptone 10-50 g/l yeast extract 2-20 g/l mineral salts 1-10 g/lbuilders 0-5 ml/l drinking water q.s. to the desired volume

A preferred example of a medium for anallergic probiotic bacterialcultures could be the following:

glucose (from maize starch) 20 g/l rice peptone 10 g/l meat peptone 10g/l yeast extract 5 g/l sodium acetate 5 g/l citrate ammonium 2 g/ldibasic potassium phosphate 2 g/l magnesium sulfate 0.1 g/l manganesesulfate 0.05 g/l tween 80 1 ml/l drinking water q.s. to the desiredvolume

The fermentation is carried out according to the teachings known to theskilled in the art and under the experimental conditions of common use.

The Applicant has verify the presence, or not, of allergen traces on aprobiotic culture grown on raw materials subject of the presentinvention.

For example, in case of milk-derived allergens, the research byanalytical way of β-lactoglobulin and lactose on the end products, withconfirmed specific and sensitive methodologies (analysis with ELISA kitspecific for the β-lactoglobulin of the type “Bovine β-lactoglubilinsELISA quantitation kit—Bethyl Laboratories”, with a threshold limit of0.05 ppm and analysis with chemoenzymatic kit and UV-vis detection forthe lactose of the type “Lactose/D-glucose—Boehringer Mannheim, cod.10986119, with a threshold limit of 7 ppm) gives a negative result and,therefore, these substances, if any, should certainly be under thedetection threshold.

At the same time, the gluten research carried out with the more refinedand, so far, more sensitive confirmed methodology (ELISA RIDASCREEN®Gliadin kit -R-Biopharm A, Darmstadt, Germany, with a sensitivity equalto 3 ppm) allows to confirm the absence of gluten. It follows that, evenif the gluten were present, its concentration should be in any caseunder the detection threshold, namely lower than 3 ppm.

The enzymatic pre-treatment on the raw materials, to be carried out ornot as a function of the requirements, is able to hydrolize milk andderivatives traces and gluten and derivatives accidentally existing inthe culture medium.

Such treatment imparts the highest safety standard for a use alsosuitable to allergic and particularly sensitive persons.

This manufacturing strategy is suitable for the probiotics productionwith an anallergic safety degree called DSS—Double Safety System.

The enzymatic pre-treatment foresees the use of at least a proteolyticenzyme and/or the use of at least a glycosidase enzyme.

In the context of the present invention, the proteolytic enzyme is ableto perform a proteolysis. The proteolytic enzyme is selected from thegroup including the proteases and/or the peptidases. The proteases andthe peptidases are selected from the group including: trypsin,chymotrypsin, pancreatin, pepsin, papain and bromelain. Preferably, theproteases and the peptidases are selected between pepsin and/orbromelain. In the context of the present invention, the glycosidaseenzyme is able to perform a hydrolityc cleavage of a glycoside. Theglycosidase enzyme is selected from the group including:alfa-glucosidase and beta-glucosidase, alfa-galactosidase andbeta-galactosidase.

Advantageously, the enzymatic treatment of the raw materials forming thegrowth broth for the probiotics is carried out with proteases (alcalasesand bromelain) and with the glycosidases.

The glycosidases are selected from the group including: lactase (orβ-galactosidase). In a preferred embodiment, the pre-treatment of theraw materials foresees the use in a sequence including three enzymes:alcalase, lactase and bromelain.

In a preferred embodiment, the selection of the enzymes and theirsequence is the following:

alcalase, which practically hydrolyzes all the proteins and particularlythose of the milk;

lactase, which hydrolyzes the lactose;

bromelain, which hydrolyzes the gluten.

The sequence shown is a function of the optimal hydrolysis pH in agradient from basic to acid; in this way, the medium preserves thenutritional properties. The alcalase, active towards theβ-lactoglobulin, the α-lactaalbumin and the caseins, allows to eliminateallergenic residuals, if any, deriving from fortuitous and unintentionalcross-contaminations with milk derivatives.

Such treatment foresees the addition to the raw materials dissolved inwater of a quantity of enzyme varying from 0.0025 and 0.0500 g/l,corresponding to 0.001-0.020 AU/l (Anson Units per Liter).

The solution is then brought to a temperature between 45 and 55° C. for15-60 minutes, with a pH between 7 and 8; preferably, a controlled pH of7.50±0.20.

The lactase, also known as β-galactosidase, is charged to the hydrolysisof the glycoside bond between glucose and galactose in the lactosedisaccharide.

The treatment with lactase is carried out following to the hydrolysiswith proteins alcalase after having brought the pH of the culture brothto a value between 6 and 7; preferably, a value of 6.50±0.20 withorganic acids (preferably lactic acid) by adding 250-2.000 NLU/l(Neutral Lactase Units per Liter), corresponding to 0.05-0.40 ml of anenzyme solution titrated at 5.000 NLU/g.

The solution is maintained at 37±5° C. for a varying period of 2-6hours. Finally, the bromelain is a proteolytic enzyme naturallycontained in the pineapple, capable of effectively hydrolysing thegliadin in fragments not recognized by the immune system and thereforenon allergenic.

The treatment is carried out by adding the fermentation medium with theenzyme to the amount of 0.005-0.010 g/l (equal to 110-220 GDU/l, GelatinDigesting Units per Liter), after correction of the pH to values of5.0-6.0 with organic acids (preferably lactic acid). The workingtemperature must be maintained at 37±5° C. for a time between 1 and 6hours.

Following to the three enzymatic treatments, it is necessary to restorethe pH at the optimal value for the fermentation of the single strains(preferably with 5N NaOH in order to basify, or with acid lactic inorder to acidify).

Next, a heat treatment for the purification of the medium is carried out(performed at temperatures between 90 and 145° C. for times varying fromfew seconds to 45 minutes), which will however denature and inactivatethe added enzymes, without further risks for the end product and theirintended people deriving from residuals of the enzyme used.

A typical industrial production design therefore foresees the followingsteps:

a. selection of the anallergic raw materials

b. dissolution of the raw materials in water

c. correction of the pH and temperature to proper values for the use ofthe proteolytic enzyme, preferably alcalase

d. addition of the enzyme and its action for the required time

e. correction of the pH and the temperature to proper values for the useof the glycolytic enzyme, preferably lactase

f. addition of the enzyme and its action for the required time

g. correction of the pH and the temperature to proper values for the useof the proteolytic enzyme, preferably bromelain

h. addition of the enzyme and its action for the required time

i. correction of the pH up to values suitable for the fermentation

j. purification through pasteurisation and/or sterilization of theculture medium.

k. cooling at the inoculum temperature typical of the probiotic strainunder production (37±2° C.).

l. inoculum of the strain.

m. fermentation

n. separation of the biomass and crioprotection

o. freeze drying.

The present invention allows then to produce anallergic probioticstrains and in particular with absolute absence of allergens, morepreferably of milk and gluten derivatives, with a wide safety of use forall the populations classes.

Advantageously, the anallergic probiotic bacterial cultures preparedaccording to the teachings of the present invention can be effectivelyused for the preparation of pharmaceutical formulations.

In view of the high number of persons allergic to the milk (3-5% of thepopulation with an age below 2 years) and the celiac persons (1% of thetotal population) it is useful to try to develop probiotic bacteriawhich can be administered also to this classes of population.

The present invention is then useful:

to the consumers, for which the transparency in the labelling isfundamental;

to the producers, which in this way can rely on a product with a totalguarantee of its anallergic properties, therefore proposable to thewhole purchasing population.

TABLE 1 International Depositary Accession Name Authority Number FilingDate Depositor 1 Streptococcus thermophilus BCCM LMG LMG P-18383 5 May1998 ANIDRAL S.R.L. 2 Streptococcus thermophilus BCCM LMG LMG P-18384 5May 1998 ANIDRAL S.R.L. 3 Lactobacillus pentosus BCCM LMG LMG P-21019 16Oct. 2001 Laboratorio Microbiologico Grana Provolone SRL Via P. Custodi12 I-28100 Novara (Italy) 4 Lactobacillus plantarum BCCM LMG LMG P-2102016 Oct. 2001 Laboratorio Microbiologico Grana Provolone SRL Via P.Custodi 12 I-28100 Novara (Italy) 5 Lactobacillus plantarum BCCM LMG LMGP-21021 16 Oct. 2001 Laboratorio Microbiologico Grana Provolone SRL ViaP. Custodi 12 I-28100 Novara (Italy) 6 Lactobacillus plantarum BCCM LMGLMG P-21022 16 Oct. 2001 Laboratorio Microbiologico Grana Provolone SRLVia P. Custodi 12 I-28100 Novara (Italy) 7 Lactobacillus plantarum BCCMLMG LMG P-21023 16 Oct. 2001 Laboratorio Microbiologico Grana ProvoloneSRL Via P. Custodi 12 I-28100 Novara (Italy) 8 Lactobacillus casei ssp.paracasei BCCM LMG LMG P-21380 31 Jan. 2002 ANIDRAL S.R.L. 9Lactobacillus belonging to the acidophilus group BCCM LMG LMG P-21381 31Jan. 2002 ANIDRAL S.R.L. 10 Bifidobacterium longum BCCM LMG LMG P-2138231 Jan. 2002 ANIDRAL S.R.L. 11 Bifidobacterium breve BCCM LMG LMGP-21383 31 Jan. 2002 ANIDRAL S.R.L. 12 Bifidobacterium lactis BCCM LMGLMG P-21384 31 Jan. 2002 ANIDRAL S.R.L. 13 Lactobacillus plantarum BCCMLMG LMG P-21385 31 Jan. 2002 MOFIN S.R.L. Via P. Custodi 12 I-28100Novara (Italy) 14 Lactococcus lactis ssp. lactis BCCM LMG LMG P-21387 15Mar. 2002 MOFIN S.R.L. Via P. Custodi 12 I-28100 Novara (Italy) 15Lactococcus lactis ssp. lactis BCCM LMG LMG P-21388 31 Jan. 2002 MOFINS.R.L. Via P. Custodi 12 I-28100 Novara (Italy) 16 Lactobacillusplantarum BCCM LMG LMG P-21389 15 Mar. 2002 MOFIN S.R.L. Via P. Custodi12 I-28100 Novara (Italy) 17 Streptococcus thermophilus DSMZ DSM 1650618 Jun. 2004 ANIDRAL S.R.L. 18 Streptococcus thermophilus DSMZ DSM 1650718 Jun. 2004 ANIDRAL S.R.L. 19 Bifidobacterium longum DSMZ DSM 16603 20Jul. 2004 ANIDRAL S.R.L. 20 Bifidobacterium breve DSMZ DSM 16604 20 Jul.2004 ANIDRAL S.R.L. 21 Lactobacillus casei ssp. rhamnosus DSMZ DSM 1660520 Jul. 2004 ANIDRAL S.R.L. 22 Lactobacillus delbrueckii ssp. bulgaricusDSMZ DSM 16606 20 Jul. 2004 ANIDRAL S.R.L. 23 Lactobacillus delbrueckiissp. bulgaricus DSMZ DSM 16607 20 Jul. 2004 ANIDRAL S.R.L. 24Streptococcus thermophilus DSMZ DSM 16590 20 Jul. 2004 ANIDRAL S.R.L. 25Streptococcus thermophilus DSMZ DSM 16591 20 Jul. 2004 ANIDRAL S.R.L. 26Streptococcus thermophilus DSMZ DSM 16592 20 Jul. 2004 ANIDRAL S.R.L. 27Streptococcus thermophilus DSMZ DSM 16593 20 Jul. 2004 ANIDRAL S.R.L. 28Bifidobacterium adolescentis DSMZ DSM 16594 21 Jul. 2004 ANIDRAL S.R.L.29 Bifidobacterium adolescentis DSMZ DSM 16595 21 Jul. 2004 ANIDRALS.R.L. 30 Bifidobacterium breve DSMZ DSM 16596 21 Jul. 2004 ANIDRALS.R.L. 31 Bifidobacterium pseudocatenulatum DSMZ DSM 16597 21 Jul. 2004ANIDRAL S.R.L. 32 Bifidobacterium pseudocatenulatum DSMZ DSM 16598 21Jul. 2004 ANIDRAL S.R.L. 33 Staphylococcus xylosus DSMZ DSM 17102 01Feb. 2005 ANIDRAL S.R.L. 34 Bifidobacterium adolescentis DSMZ DSM 1710301 Feb. 2005 ANIDRAL S.R.L. 35 Lactobacillus plantarum DSMZ DSM 17104 01Feb. 2005 ANIDRAL S.R.L. 36 Streptococcus thermophilus DSMZ DSM 17843 21Dec. 2005 ANIDRAL S.R.L. 37 Streptococcus thermophilus DSMZ DSM 17844 21Dec. 2005 ANIDRAL S.R.L. 38 Streptococcus thermophilus DSMZ DSM 17845 21Dec. 2005 ANIDRAL S.R.L. 39 Lactobacillus fermentum DSMZ DSM 18295 24May 2006 ANIDRAL S.R.L. 40 Lactobacillus fermentum DSMZ DSM 18296 24 May2006 ANIDRAL S.R.L. 41 Lactobacillus fermentum DSMZ DSM 18297 24 May2006 ANIDRAL S.R.L. 42 Lactobacillus fermentum DSMZ DSM 18298 24 May2006 ANIDRAL S.R.L. 43 Lactobacillus gasseri DSMZ DSM 18299 24 May 2006ANIDRAL S.R.L. 44 Lactobacillus gasseri DSMZ DSM 18300 24 May 2006ANIDRAL S.R.L. 45 Lactobacillus gasseri DSMZ DSM 18301 24 May 2006ANIDRAL S.R.L. 46 Lactobacillus gasseri DSMZ DSM 18302 24 May 2006ANIDRAL S.R.L. 47 Bifidobacterium adolescentis DSMZ DSM 18350 15 Jun.2006 ANIDRAL S.R.L. 48 Bifidobacterium adolescentis DSMZ DSM 18351 15Jun. 2006 ANIDRAL S.R.L. 49 Bifidobacterium adolescentis DSMZ DSM 1835215 Jun. 2006 ANIDRAL S.R.L. 50 Bifidobacterium catenulatum DSMZ DSM18353 15 Jun. 2006 ANIDRAL S.R.L.

The invention claimed is:
 1. A method for preparing a live probioticfood product or a live probiotic pharmaceutical formulation comprising(i) providing a fermentative substrate containing sulfur dioxide andsulfites at concentrations of not more than 10 mg/kg or 10 mg/literexpressed as SO₂; (ii) adding to the substrate provided in step (i) anexogenous meat peptone or an exogenous vegetal peptone selected from thegroup consisting of rice, potato, maize, chestnuts, tapioca, manioca,pea, fava beans, and mixtures thereof; (iii) fermenting one or more liveprobiotic bacteria in the fermentative substrate of step (ii) to yield aprobiotic bacterial culture containing less than 3 ppm gluten, less than7 ppm lactose, and less than 0.05 ppm beta-lactoglobulins, and (iv)preparing a live probiotic food product or live probiotic pharmaceuticalformulation comprising said probiotic bacterial culture wherein one ofthe one or more live probiotic bacteria is the Lactobaciillusacidophilus strain deposited at the BCCM LMG under deposit accessionnumber LMG P21381.
 2. The method of claim 1, wherein the fermentativesubstrate comprises (a) glucose derived from maize starch, potatostarch, beet sucrose, or cane sucrose, or (b) mono- and disaccharidesderived from complex polysaccharide hydrolysis.
 3. The method of claim1, wherein the fermentative substrate comprises glucose derived frommaize starch, potato starch, beet sucrose, or cane sucrose.
 4. Themethod of claim 1, wherein said live probiotic food product or liveprobiotic pharmaceutical formulation obtained in step (iv) is freezedried.
 5. The method of claim 1, wherein said method prepares a liveprobiotic pharmaceutical formulation.
 6. A method for preparing a liveprobiotic food product or a live probiotic pharmaceutical formulationcomprising (i) preparing a fermentative substrate comprising anexogenous meat peptone or an exogenous vegetal peptone selected from thegroup consisting of rice, potato, maize, chestnuts, tapioca, manioca,pea, fava beans, and mixtures thereof, (ii) subjecting the fermentativesubstrate to an enzymatic treatment using a proteolytic enzyme, aglycoside enzyme, or both to obtain a fermentative substrate containingsulfur dioxide and sulfites at concentrations of not more than 10 mg/kgor 10 mg/liter expressed as SO₂, wherein the proteolytic enzyme isselected from the group consisting of trypsin, chymotrypsin, pancreatin,pepsin, papain, and bromelain, and wherein the glycoside enzyme isselected from the group consisting of alpha-glucosidase andbeta-glucosidase, (iii) fermenting one or more live probiotic bacteriain the fermentative substrate to yield a probiotic bacterial culturecontaining less than 3 ppm gluten, less than 7 ppm lactose, and lessthan 0.05 ppm beta-lactoglobulins, and (iv) preparing a live probioticfood product or live probiotic pharmaceutical formulation comprising theprobiotic bacterial culture obtained in step (iii), wherein thefermentative substrate, and the live probiotic food product or liveprobiotic pharmaceutical formulation are each substantially free ofgluten and milk-derived allergens, and wherein one of the one or morelive probiotic bacteria is the Lactobaciillus acidophilus straindeposited at the BCCM LMG under deposit accession number LMG P21381. 7.The method of claim 6, wherein said enzymatic treatment comprisestreating the fermentative substrate with (a) bromelain, and (b)beta-galactosidase.
 8. The method of claim 6, wherein said enzymatictreatment comprises treating the fermentative substrate with bromelain.9. The method of claim 8, wherein (a) the fermentative substrate istreated with alcalase at 45° C.-55° C., pH 7-8, for 15-60 minutes, (b)the fermentative substrate is treated with lactase at 30° C.-40° C., pH6-7, for 2-6 hours, and (c) the fermentative substrate is treated withbromelain at 30° C.-40° C., pH 5-6, for 1-6 hours.
 10. The method ofclaim 6 wherein, following the enzymatic treatment, (a) the fermentativesubstrate pH is adjusted to a value suitable for culturing saidprobiotic bacteria; and (b) the fermentative substrate is heated to atemperature of 90° C.-145° C. for a time sufficient to inactivateenzymes used in the enzymatic treatment.
 11. The method of claim 6,wherein the fermentative substrate comprises (a) glucose derived frommaize starch, potato starch, beet sucrose, or cane sucrose, or (b) mono-and disaccharides derived from complex polysaccharide hydrolysis. 12.The method of claim 6, wherein the fermentative substrate comprises (a)glucose derived from maize starch, potato starch, beet sucrose, or canesucrose, (b) a meat peptone, and (c) a vegetal peptone selected from thegroup consisting of: rice, potato, maize, chestnuts, tapioca, manioca,pea, fava beans, and mixtures thereof.
 13. The method of claim 6,wherein said live probiotic food product or live probioticpharmaceutical formulation obtained in step (iv) is freeze dried. 14.The method of claim 6, wherein said method prepares a live probioticpharmaceutical formulation.